Phosphorus (P) is an essential nutrient for growth and development of plants, constituting up to 0.2% dry weight. Phosphorus is typically insoluble or poorly soluble in soils. Although the average P content of soils is about 0.05% (w/w), only 0.1% of the total P exists in plant accessible form (Illmer et al., 1995, Soil Biology and Biochemistry 27: 260-270). As a result, large amounts of soluble forms of P fertilizers are applied to meet the crop requirements to attain maximum production. Crops need more P than is dissolved in the soil solution to grow economically, therefore this P ‘pool’ must be replenished many times during the growing season. The ability of a soil to maintain adequate levels in the solution phase is the key to the plant available P status of a soil. However, the applied soluble forms of P fertilizers are easily precipitated into insoluble forms such as tricalcium phosphate [Ca3(PO4)2], ferric phosphate (FePO4), and aluminum phosphate (AlPO4) (Achal et al., 2007, Soil Biology and Biochemistry 39: 695-699). Inorganic P is negatively charged in most soils. Because of its particular chemistry, P reacts readily with positively charged calcium (Ca), iron (Fe) and aluminum (Al) ions to form relatively insoluble substances. When this occurs, the P is considered “fixed” or “tied up”. Thus, P is fixed in the soil by locking itself and rendered unavailable to plants. This is also the case in western Canada as the majority of western Canadian soils are neutral to alkaline pH and calcium phosphate minerals are the dominant inorganic precipitates. It has been found that approximately 75-90% of applied P fertilizer is precipitated by Ca, Fe and Al cations. These insoluble forms are not efficiently taken up by the plants and thus lead to an excess application of P fertilizer to crop fields (Khan et al., 2007, Agronomy and Sustainable Development 27: 29-43). The application of P fertilizer initially adds to the levels of available P already present in soil. A portion of applied P is used in the year of application (10-30%), and the remaining unused P reverts to forms of soil P which become increasingly less available to the plant.
As per Goldstein et al. (1993, Bio/Technology 11: 1250-1254) the unavailable phosphates built up in soils are adequate to sustain maximum crop yields globally for about 100 years. Moreover, excess P application also enhances the potential for P loss to surface waters through overland or subsurface flow that accelerates freshwater eutrophication. This is the process in bodies of water of stimulating algal growth which ultimately die and decay in the water, and deplete available oxygen. The reduced oxygen levels ultimately result in reduced higher-order aquatic plant and animal populations. The P that can contribute to the enrichment of water bodies, and hence lead to eutrophication, is a combination of P that is attached to soil particles less than 0.45 μm in size that are transported during soil movement. The risk of P losses to the environment through surface runoff is greatest on sloping lands, and where fertilizer is surface applied and then followed by rainfall or irrigation. Manitoba fresh water lakes are one such examples of eutrophication. Eutrophication of most fresh water around the world is accelerated by P inputs and therefore, P is often the limiting element, and its control is of prime importance in reducing the accelerated eutrophication of fresh waters (USDA, 2003 in Agricultural phosphorus and eutrophication, Second Edition, (Sharpley, Daniel, Sims, Lemunyon, Stevens and Parry, eds), ARS-149, pp 38).
Phosphorus is vital for stable food production systems and for buffering against climate change impacts on soil. This is important for both crop and livestock production (AIC, 2010, Agriculture Institute of Canada Notes, June 3, issue 22). It is not always realized that phosphate is a scarce raw material, probably the most critical one. High quality P reserves are diminishing and the cost of fertilizers is escalating rapidly. Global reserves of phosphate (with >20% P2O5 content) seem to be in the range of 10000 million tonnes. With a future annual consumption of 40-50 million tonnes of P2O5 these reserves would last less than 200 years (FAO, 2006, FAO (Food and Agriculture Organization) Fertilizer and Plant Nutrition Bulletin 16, pp 348). Therefore, use of phosphate fertilizers need to be as judicious as possible and nutrient use efficiency of the phosphate fertilizers are required to be improved considerably. This is a particularly relevant and important topic in the light of the increasing global population as well.
Phosphorus occurs in soil in both organic and inorganic forms that differ greatly in terms of their solubility and mobility. Phosphorus applied through mineral fertilizers is in inorganic forms of varying solubility. Even at optimal rates, the use of mineral fertilizers and organic manures can lead to a buildup of soil P over time. Plants take up inorganic phosphate in two soluble forms: monobasic (H2PO4−) and dibasic (HPO42−) ions (Vessey, 2003, Plant and Soil 255: 571-586; Banerjee et al., 2006 in Hand Book of Microbial Biofertilizers (Rai, ed) pp 137-181). Some soil microorganisms are able to solubilize these insoluble P forms through the process of organic acid production, chelation, ion exchange reactions and polymeric substances formation, and make P available to plants (Vessey, 2003; Delvasto et al., 2006, Indian J. Mar. Sci. 29: 48-51; Chang and Yang, 2009, Bioresour. Technol. 100: 1648-1658). Seed or soil inoculations with phosphate solubilizing microbes have largely been used to improve crop growth and production by solubilization of fixed and applied phosphates (Nautiyal et al., 2000, FEMS Microbiology Letters 182: 291-296; Adesemoye and Kloepper, 2009, Applied Microbiology and Biotechnology 85: 1-12). Phosphate solubilizing bacteria play a role in P nutrition by enhancing its availability to plants through release from inorganic and organic soil P pools by solubilization and mineralization. The principal mechanism in soil for mineral phosphate solubilization is the lowering of soil pH by microbial production of organic acids and mineralization of organic P by acid phosphatase enzyme (Sharma et al., 2011, J. Microbiol. Biotech. Res. 1(2): 90-95). The existence of microorganisms able to solubilize various forms of phosphates has been reported frequently elsewhere (e.g., Khan et al., 2009, J. Agric. Biol. Sci. 1(1): 48-58; Chakkaravarthy et al., 2010, J. Biol. Sci. 10(6): 531-535) but the success of utilizing the P-solubilizing plant growth promoting rhizobacteria (PGPR) as a commercial bioinoculant in different agroclimatic conditions in Canada (as well as USA) is yet to be determined appropriately. The PGPR and rhizosphere bacteria are free-living soil organisms that benefit plant growth by different mechanisms (Glick, 1995, Canadian Journal of Microbiology 41: 109-117). The ability of microorganisms to solubilize phosphorus is considered to be one of the most important traits associated with plant P nutrition (Chen et al., 2006, Applied Soil Ecology 34: 33-41). Hence, a biological seed treatment or bioinoculant with suitable formulation with naturally occurring P-solubilizing PGPR has tremendous potential to enhance production in prairie agronomic crops like canola (Brassica napus L.) with lower input cost (e.g., Banerjee and Yesmin, 2000, Agronomy Abstracts, Annual Meeting, Soil Science Society of America, pp 257). Canola is a major cash crop of the Canadian prairies (approx. 20 million acres) and any improvement in their yield potential would be substantial to the Canadian farmers as well as the economy. Thus, enhancing the production of canola with consistent performing phosphate solubilizing PGPR bioinoculant could be huge. In general, biological fertilization or biofertilizer is based on the use of natural inputs like microorganisms (e.g., bacteria, fungi) and are used to improve soil nutrient availability, produce growth stimulant for plant, improve soil stability, recycle nutrients, promote mycorrhiza symbiosis and develop bioremediation process in soil (Carvajal-Munoz and Carmona-Garcia, 2012, Livestock Research for Rural Development 24(3): pp 1-7). Hence, the naturally-occurring phosphate solubilizing PGPR could have a real potential to be used as a canola biofertilizer to enhance the canola production in western Canada and elsewhere.
U.S. Pat. No. 5,503,652 teaches the isolation of strains that are capable of promoting root elongation in plants.
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The basic requirement of profitable crop production is to produce an agronomic yield that can maximize net returns. Even the highest yield would not be of interest if its production were not cost-effective. Most farmers would like to maximize the net gains from whatever investment they can make in inputs. However, they also realize that top profits are possible only with optimal investment along with the correct decisions about the proper and innovative inputs (like P-solubilizing bioinoculant).
There has been much research conducted on the use of organisms to increase P availability in soils by “unlocking” P present in otherwise sparingly soluble forms. These microbes help in the solubilization of P from phosphate rock and other insoluble forms of soil P, and in the process decreasing their particle size, reducing it to nearly amorphous forms. Datta et al. (1982, Plant and Soil 69: 365-373) reported of a phytohormone producing phosphate solubilizing Bacillus firmus in augmenting paddy yield in acid soils of Nagaland. deFreitas et al. (1997, Biology and Fertility of Soils 24: 358-364) showed using phosphate solubilizing bacteria enhances the growth and yield of canola but P uptake in canola was not augmented. In addition to bacteria, the fungus Penicillium bilaii has been shown to increase P availability from native soils and phosphate rock sources in calcareous soils (Kucey, 1983, Canadian Journal of Soil Science 63: 671-678; Kucey and Leggett, 1989, Canadian Journal of Soil Science 69: 425-432). In fact, there is only one product in North America that contains a single action fungal P-solubilizer making it difficult for the inoculant to cope with the environmental stresses and be competitive. Bacteria are more effective in P solubilisation than fungi (Alam et al., 2002, Intl. J. Agric. Biol. 4: 454-458). Among the whole microbial population in soil, P solubilizing bacteria constitute 1-50%, while P solubilizing fungi are only 0.1-0.5% in P solubilisation potential (Chen et al., 2006, Appl. Soil Ecol. 34: 33-41). In addition, fungal inoculants are generally less competitive compared to bacterial inoculants and fungal spores are also not easy to mass-produce. However, in some other countries (such as India, Taiwan) the P-solubilizing bacteria are becoming popular (Zaidi et al., 2009, Acta Microbio Immunol Hung 56 (3): 263-284; Chang and Yang, 2009, Bioresour Technol 100 (4): 1648-1658; Ekin, 2010, African Journal of Biotechnology 9 (25): 3794-3800), ranking next in importance to the nitrogen-fixing Rhizobium inoculants, and usually more than one type of organism is used while preparing the P-solubilizing bio fertilizer. The present invention introduces not only pure culture but also a consortia based approach with multiple strains of P-solubilizing bacteria. It shows the feasibility of potential use of mixed bacteria to form synergistic consortia and will create greater competitive ability to perform consistently under different growing conditions (Yesmin and Banerjee, 2001, in Proceedings of Saskatchewan Soils and Crops Workshop 2001, pp 314-319).
Although the pure culture or consortia culture inoculant may divulge enormous possibilities for canola, for a biological inoculant to be commercially effective, it must be mass-produced efficiently and formulated into a cost-effective, uniform, and readily applicable form (Walter and Paau, 1997 in Soil Microbial Ecology: Applications in Agricultural and Environmental Management, Metting, Jr., (ed.), pp. 579-594). Much of the studies have done for identifying the possible microbiological active, yet little has been investigated on these particular aspects. The benefit of microbial inoculation for greater crop production is significantly impacted by the number of live cells introduced into soil (Duquenne et al., 1999, FEMS Microbiology Ecology 29: 331-339). Furthermore, biological activity of microbes may also decline rapidly with handling and storage procedure. Daza et al. (2000, Soil Biology and Biochemistry, 32: 567-572) evaluated a peat and a perlite-based inoculant, and showed that sucrose adhesive along with the perlite carrier gave better viability of bacteria on seeds. A key limitation to successfully commercializing beneficial microorganisms is overcoming difficulties in creating a viable, cost-effective, and user-friendly final product (Xavier et al, 2004 in Crop Management Network, Symposium Proceedings Great Plains Inoculant Forum, Saskatoon, Saskatchewan, pp. 1-6). Thus, it is critical to ascertain the length of bacterial survivability once the bacterial seed treatment is done and to attain the required level of bacterial population for the inoculant to be efficacious.
In most Canadian canola acres, treated seeds are regularly used as a critical component to control plant diseases. These fungicides/insecticides (e.g. Helix Xtra, Prosper FX, etc.) formulated as a suspension are used as seed treatments to control pre-emergence damping off, seed decay and other soil-borne diseases. It is anticipated that the bacterial cultures may not be alive with these pesticides at the recommended doses due to their high toxicity towards the bacteria (Yesmin and Banerjee, 2000, Agronomy Abstracts, Annual Meeting, Soil Science Society of America, pp 257; Yesmin and Banerjee, 2001). Our innovative approach of P-solubilizing formulations will allow high survivability of introduced bacteria across various environmental constrains, thereby ensuring higher yield and greater productivity. This will also resolve issues related to seed treatment chemicals and will give the farmers flexibility in terms of choice of seed treatment chemicals. Ensuring high survivability of these bacteria will eventually ensure greater P availability and lower input of costly P fertilizer for the crops.